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. 2006 Jun 29;25(14):3286–3297. doi: 10.1038/sj.emboj.7601212

Figure 7.

Figure 7

Daxx influences SAPK/JNK activation induced by DNA damage. (A) siRNA-induced reduction of Daxx protein. HeLa cells were transfected with pSuper-control-RNAi or pSuper-Daxx-RNAi vector. Lysates prepared at 65 h post-transfection were immunoprecipitated with anti-Daxx antibody and the immunoprecipitates were immunoblotted with anti-Daxx antibody. (B) Daxx silencing enhances cytoplasmic RASSF1C accumulation. HeLa cells were cotransfected with pSuper-control-RNAi or pSuper-Daxx-RNAi vector plus pCMV-DsRed (to indicate pSuper vector-transfected cells) and immunostained with anti-RASSF1C antibody (upper panels). The percentage of total cells that showed RASSF1C accumulation in the cytoplasm was determined by examination of 100 DsRed-positive cells/sample (lower panel). Data shown are the mean percentage±s.e.m. of data obtained from at least three independent experiments. (C) Daxx silencing promotes MMS- or UV-induced SAPK/JNK activation. HeLa cells transfected with pSuper-control-RNAi or pSuper-Daxx-RNAi vector were treated with MMS, sorbitol or UV and lysates were prepared at the indicated time points post-treatment. SAPK/JNK activity was measured by in vitro kinase assay using the GST-c-Jun substrate (lower panels). Data shown in the upper panels are the number of fold increase in P-c-Jun production in each sample compared to untreated-control. (D) The effects of Daxx silencing on MMS- or UV-induced caspase-3 activation. HeLa cells transfected with pSuper-control-RNAi or pSuper-Daxx-RNAi were treated with MMS or UV and lysates were prepared at the indicated time points post-treatment. Caspase-3 activity was measured as described in Materials and methods. Results shown are the mean fold stimulation±s.e.m. of caspase-3 activation in each sample compared to untreated-control from four independent experiments. (E) The N-terminus of Daxx inhibits MMS-induced SAPK/JNK activation. HeLa cells transfected with control, pCMV-HA-Daxx full-length, HA-Daxx1–501 or HA-DaxxC501 vector were treated with MMS and lysates were prepared at the indicated time points post-treatment. SAPK/JNK actitvity was measured as for (C) except that JNK was immunoprecipitated with anti-Flag antibody (lower panel). Data shown in upper panel are the number of fold increase in P-c-Jun production in each sample compared to untreated control.